Erythropoietin is a glycosylated protein that stimulates red blood cell production. It is produced by interstitial and capillary endotelial cells in the renal cortex and transported in the blood to the bone marrow. Koury et al., “Localization of Erythropoietin Synthesizing Cells in Murine Kidneys by in situ Hybridization,” Blood, 71:524-527 (1988); Eschbach, “The Anemia of Chronic Renal Failure: Pathophysiology and the Effects of Recombinant Erythropoietin,” Kidneys Int., 35:134-148(1989). The hormone's biological activity involves a direct receptor-mediated stimulation of the maturation and replication of late erythroid progenitor cells, proerythroblasts, and erythroblasts. Mufson et al., “Binding and Internalization of Recombinant Human Erythropoietin in Murine Erythroid Precursor Cells,” Blood, 69:1485-1490 (1987); Krantz et al., “Specific Binding of Erythropoietin to Spleen Cells Infected with the Anemia Strain of Friend Virus,” Proc. Natl. Acad. Sci. USA, 81:7574-7578 (1984). Synthesis of erythropoietin is stimulated in response to tissue hypoxia mediated by intracellular aerobic metabolism. Erslev, “Physiologic Controls of Red Cell Production,” Blood, 10:954-959 (1955). The primary protein structure of human erythropoietin includes a 27 amino acid signal peptide and a 166 amino acid mature protein. Lin et al., “Cloning and Expression of the Human Erythropoietin Gene,” Proc. Natl. Acad. Sci. USA, 82:7580-7584 (1985). Predicted molecular weight of 18.4 kDa is substantially less than the 32-kDa observed when erythropoietin is purified directly from blood or urine. The difference is due to glycosylation, three N-linked sugar chains at Asn, 24, 38, and 83, and an O-linked mucin-like moiety at Ser 126. Lai et al., “Structural Characterization of Human Erythropoietin,” J. Biol. Chem., 261:3116-3121 (1986). Compared to human, the amino acid sequences of mouse and monkey erythropoietin are 80 and 92% identical, respectively. McDonald et al., “Cloning, Sequencing, and Evolutionary Analysis of the Mouse Erythropoietin Gene,” Molecular and Cellular Biology, 6:842-848 (1986): Shoemaker et al., “Murine Erythropoietin Gene: Cloning, Expression, and Human Gene Homology,” Molecular and Cellular Biology, 6:849-858 (1986); Lin et al., “Monkey Erythropoietin Gene: Cloning, Expression and Comparison with the Human Erythropoietin Gene,” Gene, 44:201-209 (1986). The basic erythropoietin gene structure, five exons and four introns, is conserved.
Recombinant human erythropoietin (rhEPO) synthesized in Chinese Hamster Ovary (CHO) cells is produced commercially (Epogen®, Amgen, Inc. Thousand Oaks, Calif.) and widely used to support red blood cell production in people suffering from anemia secondary to chronic renal disease. Eschbach, “The Anemia of Chronic Renal Failure: Pathophysiology and the Effects of Recombinant Erythropoetin.” Kidney Int., 35:134-148 (1989); Eschbach et al., “Treatment of the Anemia of Progressive Renal Failure with Recombinant Human Erythropoietin,” N. Engl. J. Med., 321:158-163 (1989). Although the pathogenosis of the anemia is multifactorial, compensatory failure by the bone marrow to replace red blood cells largely involves a loss of functional renal tissue and a drop in endogenous erythropoietin production. Eschbach, “The Anemia of Chronic Renal Failure: Pathophysiology and the Effects of Recombinant Erythropoietin,” Kidney Int., 35:134-148 (1989); King et al., “Anemia of Chronic Renal Failure in Dogs,” J. Vet. Int. Med., 6:264-270 (1992). Synthesis of rhEPO for clinical use in restricted to eukaryotic cells due to the requirement of post-translational glycosylation for in vivo stability and bioactivity of the hormone. Takeuchi et al., “Structures and Functional Roles of the Sugar Chains of Human Erythropoietins,” Glycobiology, 1:337-346 (1991). Devoid of sugars or even the terminal sialic acid residues, erythropoietin is rapidly cleared and metabolized by the liver. Spivak et al., “The in vivo Metabolism of Recombinant Human Erythropoietin in the Rat,” Blood, 73:90-99 (1989).
Nonregenerative anemia, characterized by an inadequate production of new red blood cells, is a frequent and serious complication of kidney failure, certain types of cancer, and other chronic diseases in companion animals.
Chronic renal failure is a progressive and irreversible deterioration of kidney function that is a common and frustrating clinical problem in veterinary medicine. Although usually considered a disease of older animals, chronic renal failure is also encountered congenitally as familial renal disease (e.g., in the Norwegian elkhoud, Cocker spaniel, Samoyed, Doberman pinscher, Lhasa apso, Shih Tzu, golden retriever) (Finco, “Congenital, Inherited and Familial Renal Diseases,” In: Canine and Feline Nephrology and Urology. Osborne et al., (eds.), Baltimore: Williams & Wilkins, pages 471-483 (1995)) and in other young animals through nephrotoxic or infection mechanisms. Polzin et al., “Diseases of the Kidneys and Ureters,” In: Textbook of Veterinary Internal Medicine, Ettinger (ed), Philadelphia: WB Saunders Company. pp. 1962-2046 (1989): Krawiec. “Renal Failure in Immature Dogs.” J. Amer. Anim. Hosp. Assoc. 23:101-107 (1987). Despite a poor long-term prognosis, many dogs and cats with chronic renal failure are medically managed for years with special diets, phosphate binders, and antacids. Eventually, however, this conventional therapy fails to control the clinical signs of renal failure. Cowgill et al., “Veterinary Applications of Hemodialysis,” In: Kirk's Current Veterinary Therapy, 12th ed., Bonagura et al., (eds.), Philadelphia: W B Saunders, pages 975-977 (1995). For these animals, intermittent hemodialysis has improved survival by decreasing the uremic toxins that accumulate during renal failure. Operational dialysis units are already available in several veterinary centers across the country, and expanded use of hemodialysis in the management of renal failure in veterinary medicine is expected.
Nevertheless, even though dialysis ameliorates the uremia in canine and feline patients, lethargy, weakness, and inappetence resulting from the anemia of chronic renal failure persist. In fact, the anemia may even be compounded by blood loss in the dialyzer. Eschbach et al., “Iron Balance in Hemodialysis Patients,” Ann. Int. Med., 87:710-713 (1977). Erythropoietin treatment has become an essential component of the therapy for animals receiving hemodialysis. Even with the life-threatening risk of red cell aplasia, rhEPO is used because it represents the only erythropoietin-replacement option currently available.
Lymphosarcoma (also known as lymphoma or malignant lymphoma) is a common cancer in dogs. Although the exact cause is unknown, certain breeds including Boxer, Basset hound, St. Bernard, Scottish terrier, Airedale terrier, English bulldog, and Labrador retriever have a predisposition for development of this cancer. Nelson et al., Essentials of Small Animal Internal Medicine. St. Louis: Mosby-Year Book, Inc., pages 861-870 (1992). Treatment of lymphosarcoma consists of various chemotherapy protocols (typically utilizing vincristine, cyclophosphamide, doxorubicin, and prednosone) that result in high remission rates and allow survival for approximately 6-12 months.
Nonregenerative anemia is a common hematologic finding in dogs with lymphosarcoma. Nelson et al., Essentials of Small Animal Internal Medicine, St. Louis: Mosby-Year Book, Inc, pages 861-870 (1992); Lucroy, et al., “Anaemia Associated with Canine Lymphoma,” Comp. Haematol. Int'l 8:1-6 (1998). The anemia may be encountered during the initial diagnostic evaluation, or may develop during chemotherapy. Similarly, human cancer patients are often anemic, Miller et al., “Decreased Erythropoietin Response in Patients with the Anemia of Cancer.” N. Engl. J. Med., 322:1689-1692 (1990); Moliterrio et al., “Anemia of Cancer,” Hematol. Oncol. Clin. of N. Am., 10:345-363, (1996). Although the pathogenesis of the anemia of cancer is multifactorial, three major variables identified are: 1) the inhibition of erythropoietin production and bioactivity by inflammatory cytokines and chemotherapeutic drugs; 2) direct inhibition of erythroid progenitors by cytokines; and 3) impaired iron metabolism. Moliterrio et al., “Anemia of Cancer,” Hematol. Oncol. Clin. of N. Am., 10:345-363 (1996); Schapira et al., “Serum Erythropoietin Levels in Patients Receiving Intensive Chemotherapy and Radiotherapy,” Blood, 76:2354-2359 (1990); Means et al., “Progress in Understanding the Patheogenesis of the Anemia of Chronic Disease,” Blood, 80:1639-1647 (1992); Lacome, “Resistance to Erythropoietin.” N. Eng. J. Med., 334: 660-662 (1996); Beguin, “Erythropoietin and the Anemia of Cancer,” Acta. Clinica. Belgica, 59:36-52 (1996); Mittelman, “Anemia of Cancer: Pathogenesis and Treatment with Recombinant Erythropoietin,” Isr. J. Med. Sci., 32:1201-1206 (1996). Consistent with these etiologic variables is clinical data demonstrating that the anemia of cancer in 32-85% of human patients (depending on the cancer type) responds to pharmacologic doses of rhEPO. Mittelman, “Anemia of Cancer: Pathogenesis and Treatment with Recombinant Erythropoietin,” Isr. J. Med. Sci., 321201-1206 (1996); Spivak, “Recombinant Human Erythropoietin and the Anemia of Cancer,” Blood, 84:997-1004 (1994); Henry, “Recombinant Human Erythropoietin Treatment of Anemia Cancer Patients,” Cancer Practice, 4:180-184 (1996). Furthermore, in vitro studies demonstrate a reversal of cytokine-mediated inhibition of erythropoiesis with increased concentrations of rhEPO. Means et al., “Inhibition of Human Erythroid Colony-Forming Units by Gamma Interferon can be Corrected by Recombinant Human Erythropoietin.” Blood, 78:2564-2567 (1991). However, treatment with a safe “non-immunogenic” preparation of exogenous erythropoietin to alleviate the anemia associated with cancer and chemotherapy in dogs or cats has not been possible.
As noted above, erythropoietin therapy is often indicated for the management of nonregenerative anemia. In cases of primary erythropoietin deficiency, as in anemia secondary to chronic renal failure, erythropoietin therapy may become essential for life. The only opinion currently available to veterinarians is rhEPO, with its inherent risk of immunogenicity. Cowgill, “Erythropoietin: Its Use in the Treatment of Chronic Renal Failure in Dogs and Cats,” Proceedings of the 15th Annual Waltham/OSU Symposium for the Treatment of Small Animal Diseases. Ohio State University, pages 65-71 (1991): Giger, “Erythropoietin and Its Clinical Use,” Compend. Contin. Ed. Pract. Vet., 14:25-34 (1992); Cowgill, “Medical Management of the Anemia of Chronic Renal Failure.” In: Canine and Feline Nephrology and Urology, Osborne, et al., (eds.), Baltimore: Williams and Wilkins, pages 539-554 (1995); Cowgill et al., “Use of Recombinant Human Erythropoietin for Management of Anemia in Dogs and Cats with Renal Failure. J. Am. Vet. Med. Assoc., 212:521-528 (1998); Stokol et al., “Pure Red Cell Aplasia After Recombinant Human Erythropoietin Treatment in Normal Beagle Dogs,” Vet. Pathol., 34:474 (1997). When dogs develop red cell aplasia secondary to rhEPO, continued therapy is contraindicated for two reasons. First, the in vivo bioactivity of rhEPO is blocked, most likely because it no longer even reaches the erythroid progenitor target cells in the bone marrow. Second, the rhEPO therapy is casually associated with the red cell aplasia. Spontaneous recovery of the bone marrow is possible with cessation of the rhEPO treatments. Unfortunately, in many of the clinical cases where either the production or bioactivity of endogenous erythropoietin is compromised by the patient's primary disease, this spontaneous recovery of erythropoiesis never develops or is so inadequate that the red cell aplasia proves to be fatal.
In dogs and cats, the progressive clinical syndrome associated with chronic diseases, such as renal failure, also includes development of a nonregenerative anemia. In parallel to the human literature, studies have documented low serum concentrations of erythropoietin despite the anemia. King et al., “Anemia of Chronic Renal Failure in Dogs,” J. Vet. Int. Med., 6:264-270 (1992). Therapeutic use of rhEPO in dogs and cats with anemia secondary to chronic renal failure results in a rapid and significant red blood cell response. Cowgill, “Erythropoietin: Its Use in the Treatment of Chronic Renal Failure in Dogs and Cats.” Proceedings of the 15th Annual Waltham/OSU Symposium for the Treatment of Small Animal Diseases. Ohio State University, pages 65-71 (1991); Giger, “Erythropoietin and Its Clinical use,” Compend. Cantin. Ed. Pract. Vet., 14:14 25-34 (1992); Cowgill, “Medical Management of the Anemia of Chronic Renal Failure,” In: Canine and Feline Nephrology and Urology. Osborne et al. (eds.). Baltimore: Williams and Wilkins, pages 539-554 (1995); Cowgill et al., “Use of Recombinant Human Erythropoietin for Management of Anemia in Dogs and Cats with Renal Failure.” J. Am. Vet. Med. Assoc., 212:521-528 (1998). Depending on the dose administered, hematocrit and hemoglobin values can be restored to a normal range within several weeks and treated animals display increased alertness, physical strength, appetite, and overall attitude. These findings strongly suggest that the persistent anemia contributes significantly to some of the clinical manifestations of chronic renal failure. Unfortunately, the red blood cell status of both dogs and cats often declines in 1 to 4 months despite continued rhEPO therapy. Therapeutic failure of rhEPO in companion animals, estimated with an incidence between 20 and 50%, appears to result from interspecies variation in erythropoietin structure and the appearance of antibodies against the human protein. The ability of rhEPO to bind target receptors on erythroid progenitor cells is conserved, but the human protein is frequently recognized as foreign by the immune system of animals. Cowgill, “Erythropoietin: Its Use in the Treatment of Chronic Renal Failure in Dogs and Cats,” Proceedings of the 15th Annual Waltham/OSU Symposium for the Treatment of Small Animal Diseases. Ohio State University, pages 65-71 (1991); Giger, “Erythropoietin and Its Clinical Use,” Compend. Contin. Edg., Pract. Vet., 14:25-34 (1992); Cowgill, “Medical Management of the Anemia of Chronic Renal Failure,” In: Canine and Feline Nephrology and Urology. Osborne et al., (eds.), Baltimore: Williams and Wilkins, pages 539-554 (1995); Cowgill et al., “Use of Recombinant Human Erythropoietin for Management of Anemia in Dogs and Cats with Renal Failure,” J. Am. Vet. Med. Assoc., 212:521-528 (1998).
Anti-rhEPO antibodies are thought not only to effectively block rhEPO's bioactivity, but also have the potential to cross react with residual endogenous erythropoietin and lead to a pure red cell aplasia. This problem of immunogenicity can be life threatening and has severely limited the therapeutic potential of rhEPO for veterinary applications. The concept of erythropoietin replacement is appropriate for comparison animals, the problem is the immunogenicity of rhEPO.
The cDNA sequences for a number of mammalian erythropoietin genes are disclosed in Wen, et al., “Erythropoietin Structure-Function Relationships: High Degree of Sequence Homology Among Mammals,” Blood 82(5):1507-16 (1993). Although the nucleotide sequence for dog is disclosed, that sequence is missing the coding information for the first four codons of canine erythropoitin, which is critical for recombinant production of this protein.
The present invention is directed to overcoming the above-noted deficiencies in the prior art.